Ultra high affinity neutralizing antibodies

ABSTRACT

Ultra high affinity antibodies with binding affinities in the range of 10 10  M −1 , and even 10 11  M −1  are disclosed. Such antibodies include antibodies having novel high affinity complementarity determining regions (CDRs), especially those with framework and constant regions derived from either humans or mice. Methods of preparing and screening such antibodies, as well as methods of using them to prevent and/or treat disease, especially virus-induced diseases, are also disclosed.

[0001] This application claims priority of U.S. Provisional ApplicationSerial No. 60/178,426, filed Jan. 27, 2000, the disclosure of which ishereby incorporated by reference in its entirety.

BACKGROUND OF THE INVENTION

[0002] The present invention relates to novel ultra high affinityneutralizing antibodies.

[0003] The current incidence of infection caused by resistant ordifficult to control microbes has created a need for newer approaches tocontrolling such organisms, as well as to treating those alreadyinfected.

[0004] Among the more difficult infectious agents to control and treatare the viruses. For example, respiratory syncytial virus (RSV) is themajor cause of acute respiratory illness in young children admitted tohospitals and the major cause of lower respiratory tract infection inyoung children. A major obstacle to producing an effective vaccineagainst such agents as RSV has been the issue of safety. Conversely, theuse of immunoglobulins against such viral agents has proven of somevalue. For example, studies have shown that high-titred RSVimmunoglobulin was effective both in prophylaxis and therapy for RSVinfections in animal models.

[0005] An alternative approach has been the development of antibodies,especially neutralizing monoclonal antibodies, with high specificneutralizing activity. One drawback to this route has been the need toproduce human antibodies rather than those of mouse or rat and thusminimize the development of human anti-mouse or anti-rat antibodyresponses, which potentially results in further immune pathology.

[0006] An alternative approach has been the production of human-murinechimeric antibodies in which the genes encoding the mouse heavy andlight chain variable regions have been coupled to the genes for humanheavy and light chain constant regions to produce chimeric, or hybrid,antibodies.

[0007] In some cases, mouse CDRs have been grafted onto human constantand framework regions with some of the mouse framework amino acids beingsubstituted for correspondingly positioned human amino acids to providea “humanized” antibody. [Queen, U.S. Pat. Nos. 5,693,761 and 5,693,762].However, such antibodies contain intact mouse CDR regions and have metwith mixed effectiveness, producing affinities often no higher than 10⁷to 10⁸ M⁻¹ .

[0008] A humanized anti-RSV antibody with good affinity has beenprepared and is currently being marketed. [See: Johnson, U.S. Pat. No.5,824,307]

[0009] The production of ultra high affinity antibodies would bedesirable from the point of view of both the neutralizing ability ofsuch an antibody as well as from the more practical aspects of needingto produce less antibody in order to achieve a desirable degree ofclinical effectiveness, thereby cutting costs of production and/orallowing a greater degree of clinical effectiveness for administrationin the same volume.

BRIEF SUMMARY OF THE INVENTION

[0010] The present invention relates to high affinity neutralizingantibodies and active fragments thereof exhibiting affinity constants ofat least 10¹⁰ M⁻¹, and even 10¹¹ M⁻¹, and more specifically to such aneutralizing monoclonal immunoglobulin, including antibodies and/oractive fragments thereof, wherein the antibody and/or fragment has humanconstant regions.

[0011] The present invention solves the above-mentioned problems byproviding high affinity neutralizing antibodies without the presence ofintact mouse CDR regions that cause human anti-mouse antibody reactions(HAMA) and with sufficiently high affinity neutralizing activity toreduce cost and efficacy of overall production.

[0012] One aspect of the present invention relates to high affinityneutralizing antibodies with affinity constants of at least 10¹⁰ M⁻¹,and even 10¹¹ M⁻¹, and with specificity towards specific antigenicdeterminants, such as those exhibited by virus-expressed proteins.

[0013] One object of the present invention is to provide such highaffinity neutralizing antibodies with specificity toward antigensproduced by viruses, such as where such antigens are expressed byvirus-infected cells in a mammal, especially a human.

[0014] In one such embodiment, the high affinity neutralizingimmunoglobulin, including antibodies and/or active fragments thereof, ofthe present invention, and active fragments thereof, are specific forrespiratory syncytial virus (RSV), most especially for the F antigenexpressed by said RSV and also expressed on the surfaces of cellsinfected with RSV (the presence of which antigen on the cell surfacecauses fusion of the cells into syncytia),

[0015] Thus, in one embodiment, a high affinity neutralizingimmunoglobulin, including antibodies and/or active fragments thereof, ofthe present invention binds to the same epitope on RSV as the antibodywhose light chain variable chain has the sequence of SEQ ID NO: 1 (shownin FIG. 1A) and whose heavy chain variable chain has the sequence of SEQID NO: 2 (shown in FIG. 1B).

[0016] It is an object of the present invention to provide ultra highaffinity neutralizing antibodies having substantially the frameworkregions of the immunoglobulin disclosed in FIG. 1 (with the samespecificity of that immunoglobulin, which is an anti-RSV antibodystructure) but wherein the immunoglobulins, including antibodies andactive fragments thereof, of the present invention contain one or moreCDRs (complementarity determining regions) whose amino acid sequencesare independent of those in the so-called reference antibody, althoughsaid sequences may, in some cases, differ by no more than one amino acidand this may be limited to a difference in only one of said CDR regions.

[0017] In a preferred embodiment of the present invention, the novelimmunoglobulins of the present invention will differ from the antibodyof FIG. 1 (hereafter, the “basic antibody” or “reference antibody” or“reference immunoglobulin”) only in the sequences of one or more of theCDRs and, in a most preferred embodiment these differences occur only inCDRs L2, L3, H1, and H3.

[0018] Especially preferred embodiments of the present invention havethe framework sequences depicted in FIG. 1, thus having the heavy andlight chain variable sequences depicted in FIGS. 3, 4, 5, 6, and 7.

[0019] In one embodiment, the high affinity neutralizing antibodies ofthe invention include a human constant region.

[0020] In a preferred embodiment, a high affinity RSV-neutralizingantibody of the invention, including active fragments thereof, with anaffinity constant (K_(a)) of at least as high as 10¹⁰ M⁻¹, and even 10¹¹M⁻¹, is a recombinant immunoglobulin, such as an antibody or activefragment thereof, that includes a human constant region and frameworkregions for the heavy and light chains wherein at least a portion of theframework is derived from a human antibody (or from a consensus sequenceof a human antibody framework), an example of said framework regionsdepicted for the antibody sequences of FIG. 1.

[0021] In one embodiment, all of the framework is derived from a humanantibody (or a human consensus sequence).

[0022] In another highly specific embodiment, a high affinityRSV-neutralizing antibody, with an affinity of at least 10¹⁰ M⁻¹, is arecombinant antibody having a human constant region, one or more CDRsthat are derived from a non-human antibody in which at least one of theamino acids in at least one of the CDRs is changed and in which all or aportion of the framework is derived from a human antibody (or aconsensus sequence of a human antibody framework).

[0023] In a separate embodiment, a humanized neutralizing immunoglobulinthat binds to the same epitope as the basic or reference antibody orimmunoglobulin whose variable chains are shown in FIG. 1, and that hasan affinity of at least 10¹¹ M⁻¹, includes at least one of the followingamino acids at the following positions of the CDRs: an alanine atposition 2 of CDR H1, a phenylalanine at position 6 of CDR H3, aphenylalanine at position 3 of CDR L2, and a phenylalanine at position 5of CDR L3. Other embodiments comprise other single amino acidsubstitutions at these locations.

[0024] It is another object of the present invention to providecompositions comprising the immunoglobulins disclosed herein whereinsaid structures are suspended in a pharmacologically acceptable diluentor excipient.

[0025] It is a still further object of the present invention to providemethods of preventing and/or treating respiratory syncytial viruscomprising the administering to a patient at risk thereof, or afflictedtherewith, of a therapeutically effective amount of a compositioncontaining an immunoglobulin of the invention, such as where saidantibodies or active fragments thereof exhibit the specificity andaffinity properties disclosed herein for the immunoglobulins of theinvention.

Definitions

[0026] The term “antigen” refers to a structure, often a polypeptide orprotein, present on the surface of a microorganism, such as a virus, forwhich an antibody has affinity and specificity.

[0027] The term “antigenic determinant” refers to a specific bindingsite on an antigenic structure for which an immunoglobulin, such as anantibody, has specificity and affinity. Thus, a particle, such as avirus, may represent an antigen but may have on its surface a number ofseparate, and different, antigenic sites such as where the virus has anumber of different surface proteins and each represents a distinctpotential binding site for an immunoglobulin.

[0028] The term “immunoglobulin” refers to a protein or polypeptidehaving specificity and affinity for an antigen or antigenic determinant.This term includes antibodies, commonly depicted as tetrameric, as wellas active fragments thereof, such fragments having specificity andaffinity for antigens or antigenic determinants. Thus, “immunoglobulin”as used herein includes antibodies and all active fragments thereof.

[0029] The term “antibody” refers to a protein or polypeptide havingaffinity for an antigenic determinant, usually one found on the surfaceof a microorganism, especially a virus. Such an antibody is commonlycomposed of 4 chains and is thus tetrameric.

[0030] The term “neutralizing immunoglobulin” or “neutralizing antibody”refers to the ability of the immunoglobulins, including antibodies, ofthe present invention to reduce the replication of microorganisms,especially viruses, in organisms as well as in cell cultures. Anindication of such ability is the data from the microneutralizationassays disclosed hereinbelow. Such a structure usually has both variableand constant regions whereby the variable regions are mostly responsiblefor determining the specificity of the antibody and will comprisecomplementarity determining regions (CDRs).

[0031] The term “complementarity determining region” or “CDR” refers tovariable regions of either H (heavy) or L (light) chains contains theamino acid sequences capable of specifically binding to antigenictargets. These CDR regions account for the basic specificity of theantibody for a particular antigenic determinant structure. Such regionsare also referred to as “hypervariable regions.”

[0032] The term “active fragment” refers to a portion of an antibodythat by itself has high affinity for an antigenic determinant andcontains one or more CDRs accounting for such specificity. Non-limitingexamples include Fab, F(ab)′₂, heavy-light chain dimers, and singlechain structures, such as a complete light chain or complete heavychain.

[0033] The term “specificity” refers to the ability of an antibody tobind preferentially to one antigenic site versus a different antigenicsite and does not necessarily imply high affinity.

[0034] The term “affinity” refers to the degree to which an antibodybinds to an antigen so as to shift the equilibrium of antigen andantibody toward the presence of a complex formed by their binding. Thus,where an antigen and antibody are combined in relatively equalconcentration, an antibody of high affinity will bind to the availableantigen so as to shift the equilibrium toward high concentration of theresulting complex.

[0035] The term “affinity constant” refers to an association constantused to measure the affinity of an antibody for an antigen. The higherthe affinity constant the greater the affinity of the immunoglobulin forthe antigen or antigenic determinant and vice versa. An affinityconstant is a binding constant in units of reciprocal molarity. Such aconstant is readily calculated from the rate constants for theassociation-dissociation reactions as measured by standard kineticmethodology for antibody reactions.

BRIEF DESCRIPTION OF THE DRAWINGS

[0036]FIG. 1 shows the amino acid sequence of the light and heavy chainvariable regions of an anti-RSV antibody wherein the CDR regions areunderlined while non-underlined residues form the framework regions ofthe variable regions of each chain. In this antibody, the CDRs arederived from a mouse anti-RSV antibody while the framework regionsconsist mostly of sequences derived from a human antibody. For each CDR,locations at which amino acid replacements were used to achieve the highaffinity CDRs and antibodies disclosed herein are in bold face. Inaccordance with the disclosure herein, such replacements were only inCDRs L2, L3, H1 and H3. FIG. 1A shows the light chain variable regionand FIG. 1B shows the heavy chain variable region of the light and heavychains, respectively. Constant region sequences are not shown. Thesesequences are present in the basic clone (see Table 2), designatedIX-493 throughout this disclosure (i.e., SWSG—meaning a serine (S) atthe key position (see tables 1 and 3) of high affinity CDR H1, atryptophan (W) at the key position of high affinity CDR H3, a serine (S)at the key position of high affinity CDR L2, and a glycine (G) at thekey position of high affinity CDR L3). For purposes of this disclosure,this is the “reference antibody.”

[0037]FIG. 2 shows affinity comparisons for a particular set ofbeneficial or high affinity clones. The clonal designations are on theleft of the legend at the right of the drawing along with the indicatedsubstitutions at CDRs H1, H3, L2, and L3 shown on the right of thelegend. Measurements are by ELISA (OD at 560 nm shown on the left axis).Clone L1FR represents the results for the reference antibody structureof FIG. 1.

[0038]FIG. 3 shows the heavy and light chain variable regions for thepreferred embodiment of clone 1 (Table 2) of the invention disclosedherein. CDR regions are underlined while the amino acid differencesversus the antibody of FIG. 1 are indicated in bold face. Thus, thispreferred (i.e., high affinity) antibody has several of the highaffinity CDRs (Table 3) present which give rise to higher affinity (over10¹⁰ M⁻¹) than the basic or reference antibody.

[0039]FIG. 4 shows the heavy and light chain variable regions for thepreferred embodiment of clone 2 (Table 2) of the invention disclosedherein. CDR regions are underlined while the amino acid differencesversus the antibody of FIG. 1 are indicated in bold face. Thus, thispreferred (i.e., high affinity) antibody has several of the highaffinity CDRs (Table 3) present which give rise to higher affinity (over10¹⁰ M⁻¹) than the basic or reference antibody.

[0040]FIG. 5 shows the heavy and light chain variable regions for thepreferred embodiment of clone 3 (Table 2) of the invention disclosedherein. CDR regions are underlined while the amino acid differencesversus the antibody of FIG. 1 are indicated in bold face. Thus, thispreferred (i.e., high affinity) antibody has several of the highaffinity CDRs (Table 3) present which give rise to higher affinity (over10¹⁰ M⁻¹) than the basic or reference antibody.

[0041]FIG. 6 shows the heavy and light chain variable regions for themost preferred embodiment of clone 22 (Table 4) of the inventiondisclosed herein. CDR regions are underlined while the amino acidchanges versus the antibody of FIG. 1 are indicated in bold face. Thus,this most preferred (i.e., highest affinity) antibody has several of thehigh affinity CDRs (Table 3) present which give rise to higher affinity(over 10¹¹ M⁻¹) than the basic or reference antibody).

[0042]FIG. 7 shows the heavy and light chain variable regions for thepreferred embodiment of clone 23 (Table 4) of the invention disclosedherein. CDR regions are underlined while the amino acid changes versusthe antibody of FIG. 1 are indicated in bold face. Thus, this preferred(i.e., highest affinity antibody, has several of the high affinity CDRs(Table 3) present which give rise to higher affinity (over 10¹¹ M⁻¹)than the basic or reference antibody).

[0043]FIG. 8 shows the results of microneutralization experiments onseveral of the ultra-high affinity antibody clones disclosed herein. Theamino acids present at the key positions of the high affinitycomplementarity determining regions (see Table 3) are shown at the rightin the order H1, H3, L2, and L3 (as also shown in Table 2 where theclones are simply numbered but the table compares to this figure byrelying on the actual amino acids used at the critical positions asdisclosed in the Table and in the legend at the right of this figure).Thus, for clone 4D2-7, there is an alanine (A) at the key position ofhigh affinity CDR H1, a phenylalanine (F) at the key position of highaffinity CDR H3, a phenylalanine (F) at the key position of highaffinity CDR L2, and a phenylalanine (F) at the key position of highaffinity CDR L3. Briefly, about 25,000 HEp-2 cells were added to each ofthe wells of a 96 well plate along with RSV and a given concentration ofthe antibody (antibody concentration per well is shown on theabscissa—See Example 2 for exact details). After 5 days growth, thecells were fixed, treated with biotinylated anti-F MAb, then bound toavidin-peroxidase complex and the ability of the bound peroxidase toreact thionitrobenzoic acid was determined by measuring O.D. at 450 nm.The amount of F protein present was an indicator of the extent of viralreplication thereby resulting in more reaction of substrate byperoxidase and increased absorption. Thus, the lower the OD 450 value,the greater the neutralizing ability of the indicated concentration ofthe given antibody. Here, IX-493 (SWSG—meaning a serine (S) at the keyposition of high affinity CDR H1, a tryptophan (W) at the key positionof high affinity CDR H3, a serine (S) at the key position of highaffinity CDR L2, and a glycine (G) at the key position of high affinityCDR L3) is the “reference antibody” (also denoted L1FR and IX493L1FR).

[0044]FIG. 9 shows results for microneutralization assays of several ofthe antibodies disclosed herein but where only Fab fragments wereemployed for neutralization of antibody replication. Here, IX-493 (SWSG)is the reference Fab fragment and is derived from antibody Medi-493(see: Johnson et al (1997)—ref. 23).

[0045]FIG. 10 shows the results of microneutralization for an antibodyspecific for RSV as compared to similar experiments for Fab fragments ofthe same antibody. Here, Medi 493 represents the antibody while IX-493LIFR represents the Fab fragment of this antibody. The other lines arefor Fab fragments of several of the antibodies produced according to thepresent invention (given various letter-digit code designations forinternal identification but having nothing to do with their relativeefficacy as neutralizing antibodies).

DETAILED DESCRIPTION OF THE INVENTION

[0046] The present invention is directed to ultra high affinityneutralizing antibodies and active fragments thereof having affinityconstants of at least 10¹⁰ M⁻¹. Active fragments of these antibodies arefragments containing at least one high affinity complementaritydetermining region (CDR).

[0047] With the advent of methods of molecular biology and recombinanttechnology, it is now possible to produce antibody molecules byrecombinant means and thereby generate gene sequences that code forspecific amino acid sequences found in the polypeptide structure of theantibodies. Such antibodies can be produced by either cloning the genesequences encoding the polypeptide chains of said antibodies or bydirect synthesis of said polypeptide chains, with in vitro assembly ofthe synthesized chains to form active tetrameric (H₂L₂) structures withaffinity for specific epitopes and antigenic determinants. This haspermitted the ready production of antibodies having sequencescharacteristic of neutralizing antibodies from different species andsources.

[0048] Regardless of the source of the immunoglobulins, or how they arerecombinantly constructed, or how they are synthesized, in vitro or invivo, using transgenic animals, such as cows, goats and sheep, usinglarge cell cultures of laboratory or commercial size, in bioreactors orby direct chemical synthesis employing no living organisms at any stageof the process, all immunoglobulins have a similar overall 3 dimensionalstructure. In the case of an antibody, this structure is often given asH₂L₂ and refers to the fact that antibodies commonly comprise 2 light(L) amino acid chains and 2 heavy (H) amino acid chains. Both chainshave regions capable of interacting with a structurally complementaryantigenic target. The regions interacting with the target are referredto as “variable” or “V” regions and are characterized by differences inamino acid sequence from antibodies of different antigenic specificity.

[0049] The variable region of either H or L chains contains the aminoacid sequences capable of specifically binding to antigenic targets.Within these sequences are smaller sequences dubbed “hypervariable”because of their extreme variability between antibodies or activefragments of differing specificity. Such hypervariable regions are alsoreferred to as “complementarity determining regions” or “CDR” regions.These CDR regions account for the basic specificity of the antibody fora particular antigenic determinant structure.

[0050] The CDRs represent non-contiguous stretches of amino acids withinthe variable regions but, regardless of species, the positionallocations of these critical amino acid sequences within the variableheavy and light chain regions have been found to have similar locationswithin the amino acid sequences of the variable chains. The variableheavy and light chains of all canonical antibodies each have 3 CDRregions, each non-contiguous with the others (termed L1, L2, L3, H1, H2,H3) for the respective light (L) and heavy (H) chains. The accepted CDRregions have been described by Kabat et al, J. Biol. Chem. 252:6609-6616(1977). The numbering scheme is shown in the figures, where the CDRs areunderlined and the numbers follow the Kabat scheme.

[0051] In all mammalian species, antibody polypeptides contain constant(i.e., highly conserved) and variable regions, and, within the latter,there are the CDRs and the so-called “framework regions” made up ofamino acid sequences within the variable region of the heavy or lightchain but outside the CDRs.

[0052] The immunoglobulins disclosed according to the present inventionafford extremely high affinity (on the order of 10¹⁰ M⁻¹, and even 10¹¹M⁻¹, for the affinity constant, or K_(a), defined as an associationconstant describing the binding of antibody and antigen as the ligands)for epitopes, or antigenic determinants, found in macromolecules,especially proteins, and most especially proteins expressed by virusesand other microorganisms, such as proteins expressed on the surfaces ofviruses as well as the surfaces of cells infected with a virus. The highaffinity antibodies of the present invention are neutralizing antibodiesand thus reduce the replication of viruses in organisms as well as incell cultures while maintaining sufficient homology to the antibodyamino acid sequences of the recipient so as to prevent adverseimmunological pathology. The latter feature is achieved through the useof constant regions similar to those of the recipient organism, such asa mammal and most especially a human. This feature is also achievedthrough the use of framework amino acid sequences similar, if notidentical, to those found in antibodies from the recipient organism. Inthe latter case, some amino acid replacements may be made in theframework sequences so as to facilitate and maintain the high affinityinteraction between the novel CDRs of the present invention and theantigen for which said antibodies show specificity.

[0053] As used herein, terms such as “antibody” and “active fragment” or“fragment” are not to be considered limiting in determining the fullextent of the present invention. Thus, the fact that the term “antibody”is used rather than “active fragment” or “immunoglobulin” is not to betaken as a limitation on the invention or its uses so long as therequisite properties of specificity and affinity are exhibited by saidstructure.

[0054] In accordance with the invention disclosed herein, the affinityconstants characterizing the affinities of the high affinityneutralizing antibodies, and active fragments thereof, of the presentinvention are association constants and were measured by the kinetics ofantigen-antibody complex formation, with the rate constant forassociation to form the complex being denoted as k_(assoc) or k_(on) andthe dissociation constant being denoted as k_(diss) or k_(off).Measurement of such constants is well within the ordinary skill in theart and the details will not be described further herein except forgeneral methodology and specific conditions, where appropriate, asrecited in the examples given herein to further describe the invention.

[0055] The high affinity antibodies of the present invention can beachieved, as already described, through genetically engineeringappropriate antibody gene sequences, i.e., amino acid sequences, byarranging the appropriate nucleotide sequences and expressing these in asuitable cell line. Any desired nucleotide sequences can be producedusing the method of codon based mutagenesis, as described, for example,in U.S. Pat. Nos. 5,264,563 and 5,523,388. Such procedures permit theproduction of any and all frequencies of amino acid residues at anydesired codon positions within an oligonucleotide. This can includecompletely random substitutions of any of the 20 amino acids at andesired position or in any specific subset of these. Alternatively, thisprocess can be carried out so as to achieve a particular amino acid adesired location within an amino acid chain, such as the novel CDRsequences according to the invention. In sum, the appropriate nucleotidesequence to express any amino acid sequence desired can be readilyachieved and using such procedures the novel CDR sequences of thepresent invention can be reproduced. This results in the ability tosynthesize polypeptides, such as antibodies, with any desired amino acidsequences. For example, it is now possible to determine the amino acidsequences of any desired domains of an antibody of choice and,optionally, to prepare homologous chains with one or more amino acidsreplaced by other desired amino acids, so as to give a range ofsubstituted analogs.

[0056] In applying such methods, it is to be appreciated that due to thedegeneracy of the genetic code, such methods as random oligonucleotidesynthesis and partial degenerate oligonucleotide synthesis willincorporate redundancies for codons specifying a particular amino acidresidue at a particular position, although such methods can be used toprovide a master set of all possible amino acid sequences and screenthese for optimal function as antibody structures or for other purposes.Such methods are described in Cwirla et al, Proc. Natl. Acad. Sci.87:6378-6382 (1990) and Devlin et al., Science 249:404-406 (1990).

[0057] In accordance with the invention disclosed herein, enhancedantibody variants can be generated by combining in a single polypeptidestructure one, two or more novel CDR sequences according to theinvention, each shown to independently result in enhanced bindingactivity. In this manner, several novel amino acids sequences can becombined into one antibody, in the same or different CDRs, to producehigh affinity antibodies within the present invention. For example, 3such novel CDR sequences may be employed and the resulting antibodiesscreened for affinity for a particular antigenic structure, such as theF antigen or RSV. The overall result would thus be an iterative processof combining various single amino acid substitutions and screening theresulting antibodies for antigenic affinity in a step by step manner.Such methods were used to prepare some of the antibodies embodied withinthe present invention. Such methods also represent a convenient, iftedious, means of optimizing the antibody sequences of the presentinvention, especially the sequences of the CDR regions of saidantibodies.

[0058] Using the novel sequences and methods of the present invention,such an approach avoids the time and expense of generating and screeningall possible permutations and combinations of antibody structure in aneffort to find the antibody with the maximum efficiency. Conversely,complete randomization of a single 10 amino acid residue CDR wouldgenerate over 10 trillion variants, a number virtually impossible toscreen.

[0059] This iterative method can be used to generate double and tripleamino acid replacements in a stepwise process so as to narrow the searchfor antibodies having higher affinity.

[0060] Conversely, it should be recognized that not all locations withinthe sequences of the different antibody domains may be equal.Substitutions of any kind in a particular location may be helpful ordetrimental. In addition, substitutions of certain kinds of amino acidsat certain locations may likewise be a plus or a minus as it affectsaffinity for the particular antigen. For example, it may not benecessary to try all possible hydrophobic amino acids at a givenposition. It may be that any hydrophobic amino acid will do as well.Alternatively, an acidic or basic amino acid at a given location mayprovide large swings in measured affinity. It is therefore necessaryalso to learn the “rules” of making such substitutions but thedetermination of such “rules” may not require the study of all possiblecombinations and substitutions—trends may become apparent afterexamining fewer than the maximum number of substitutions.

[0061] In accordance with the foregoing, the antibodies of the presentinvention are ultra high affinity neutralizing antibodies, such asanti-RSV antibodies, and in the latter case most preferably antibodieswith specificity toward the same epitope of RSV as the antibody of U.S.Pat. No. 5,824,307.

[0062] In addition, the affinities of the ultra high affinity antibodiesof the invention typically are at least 10¹⁰ M⁻¹. Because such highaffinities are not easily measured, except by the procedures describedherein, such value may commonly be considered as part of a range andmay, for example, be within 2 fold of 10¹⁰ M⁻¹ or be greater than 10¹⁰M−1 or may even be numerically equal to 10¹⁰ M⁻¹. In such cases, theaffinity is denoted by an affinity constant, which is in the nature of abinding constant so as to give units of reciprocal molarity. As such,the affinity of the antibody for antigen is proportional to the value ofthis constant (i.e., the higher the constant, the greater the affinity).Such a constant is readily calculated from the rate constants for theassociation-dissociation reactions as measured by standard kineticmethodology for antibody reactions.

[0063] In a specific embodiment, the high affinity neutralizingantibodies of the present invention, and active fragments thereof, haveaffinity constants for their respective antigens of at least at least10¹¹ M⁻¹, in some cases being in excess of this value, a range at thevery upper region of measurability.

[0064] The high affinity neutralizing antibodies of the presentinvention, and active fragments thereof, may be advantageously directedto any antigenic determinants desired, such as epitopes present on anytype of macromolecule, especially peptide epitopes present as part ofthe three dimensional structure of protein and polypeptide molecules.Such peptide epitopes may commonly be present on the surfaces, orotherwise be part of the structure of, microorganisms and cells, such ascancer cells. Microorganisms expressing such peptide epitopes includesbacteria and viruses. In the latter case, the proteins and polypeptidesexhibiting peptide epitopes may be molecules expressed on the surfacesof the virus or may be expressed by cells infected with a virus. Forpurposes of evaluating the efficacy of the rules and methods disclosedaccording to the present invention, a virus was chosen as one availableantigenic source for developing antibodies within the present invention.The virus chosen for further analysis was the respiratory syncytialvirus (RSV). This latter virus was chosen because it is wellcharacterized with respect to its replicative cycle as well as withrespect to the antigenic determinants found on its surface. In addition,antigens known to be expressed by cells infected with this virus arewell characterized. Thus, the virus exhibits both a surface G antigenand a surface F antigen, both proteins. The G antigen facilitatesbinding of the virus to cell surfaces and the F proteins facilitates thefusion of the virus with cells. The cells so infected also express the Fantigen on their surfaces and the latter result induces fusion of thecells to form a syncytium, hence the name of the virus. In addition,this virus is a convenient subject for analysis in that cell linesreadily infected by this virus are well known and well characterized,thereby making it an easy virus to grow and culture in vitro. Further,antibodies available for treatment of this virus are known and arecommercially available (for example, the antibodies disclosed in U.S.Pat. No. 5,824,307). For these reasons, the antibody disclosed in saidpatent, the disclosure of which patent is hereby incorporated byreference in its entirety, contains the amino acid sequences of the“reference antibody” disclosed in FIG. 1 and whose CDR sequences aresummarized in Table 1. Thus, the availability of a commerciallysuccessful antibody product as well as the well characterized propertiesof RSV made this an ideal combination to use in testing and optimizingthe antibodies of the present invention and the rules disclosed hereinfor producing high affinity CDRs for use in constructing suchantibodies. Otherwise, the antibodies disclosed herein, as well as themethods disclosed herein for preparing such antibodies, are in no waylimited to RSV as the antigen but are readily and advantageously appliedgenerally to the field of antibody technology. In addition, even thespecific embodiments provided by the present disclosure and which aredirected specifically to antigenic determinants expressed by RSV, andcells infected with RSV, also may have high affinity for other epitopes,especially those present on related viruses. Therefore, as disclosed inaccordance with the present invention, RSV, and the antibody withvariable sequences as shown in FIG. 1, are merely a convenient modelsystem used as a reference point for developing and applying the methodsof antibody technology taught by the present invention.

[0065] A high affinity neutralizing antibody according to the presentinvention, including active fragments thereof, comprises at least onehigh affinity complementarity determining region (CDR) wherein said CDRhas an amino acid sequence selected to result in an antibody having anaffinity constant (K_(a)) of at least 10¹⁰M⁻¹. In preferred embodiments,such antibody, or active fragment, comprises at least 2 high affinityCDRs, or at least 3 high affinity CDRs or even at least 4 high affinityCDRs. In highly preferred embodiments, such antibodies or activefragments comprise 3 or 4 high affinity CDRs. In one preferredembodiment, such active fragment is an Fab fragment.

[0066] The high affinity antibodies of the present invention commonlycomprise a mammalian, preferably a human, constant region and a variableregion, said variable region comprising heavy and light chain frameworkregions and heavy and light chain CDRs, wherein the heavy and lightchain framework regions are derived from a mammalian antibody,preferably a human antibody, and wherein the CDRs are derived from anantibody of some species other than a human, preferably a mouse. Wherethe framework amino acids are also derived from a non-human, the latteris preferably a mouse.

[0067] In addition, high affinity antibodies of the invention bind thesame epitope as the antibody from which the CDRs are derived, andwherein at least one of the CDRs of said ultra high affinity antibodycontains amino acid substitutions, and wherein said substitutionscomprise the replacement of one or more amino acids in the CDR regionsby non-identical amino acids, preferably the amino acids of thecorrespondingly aligned positions of the CDR regions of the humanantibody contributing the framework and constant domains.

[0068] The high affinity CDRs may be produced by amino acidsubstitutions in non-high affinity CDRs to produce such high affinityCDRs or such high affinity CDRs may be synthesized directly to form suchhigh affinity CDRs. Thus, the ultra high affinity neutralizingantibodies of the present invention may have amino acid substitutions inonly one of the CDR regions, preferably more than one CDR region, andmost preferably 3 or even 4 such regions, with possibly as many as 5 oreven all 6 of the CDRs containing at least one substituted amino acid.

[0069] In applying the methods disclosed herein to produce antibodies ofthe present invention, the method of preparing the antibodies is not alimiting factor. Thus, the high affinity neutralizing antibodies of thepresent invention may be prepared by generating polynucleotide sequencescoding for the polypeptides of the antibodies and using vectors toinsert said polynucleotide sequences into permissive cells capable ofnot only expressing such polypeptides but also of assembling them intocharacteristic tetrameric antibody structures that are then retrievedfrom the cells or cell cultures, possibly being secreted into the mediumby such cells. Technologies for such manufacturing procedures arealready known and patented and are not essential to practicing thepresent invention. [see: Morrison et al, U.S. Pat. No. 5,807,715]. Inaddition, the polypeptide chains of the antibodies of the presentinvention may be synthesized chemically, with or without the addition ofenzymes, and then chemically joined to form tetrameric structures of theusual H₂L₂ configuration. Thus, any method of preparing the antibodiesdisclosed herein can be utilized.

[0070] The present invention is also directed to the formation of highaffinity neutralizing antibodies, with the properties alreadyenumerated, having high affinity as a result predominantly of havinghigh affinity CDR sequences. In accordance with the present invention,the CDR sequences of the antibodies disclosed herein have been optimizedso as to confer upon the antibody molecule the ultra high affinitiescharacteristic of the antibodies of the present invention. Such CDRsequences, together with the framework sequences disclosed herein,especially those taught by the sequences of FIGS. 1, 3, 4, 5, 6, and 7,and most especially when used with constant region sequencescharacteristic of the antibodies of the organism acting as recipient ofthe antibodies of the present invention when used therapeutically,produce the antibodies of the present invention in their more specificembodiments. However, the methods of the present invention are morespecifically directed to the CDR amino acid sequences.

[0071] To produce immunoglobulins, such as antibodies and/or activefragments thereof, within the present invention, e.g., high affinityneutralizing antibodies, the rules taught by the present invention areused advantageously to produce antibody molecules whose structuresincorporate the sequences of the high affinity CDR sequences disclosedaccording to the present invention. Thus, the high affinity neutralizingantibodies of the present invention are, in essence, not truly“monoclonal” antibodies as that term is commonly used, since they do nothave to be produced by cloning anything. As already mentioned, thesequences of the antibodies of the invention may be directly synthesizedand thus may not be identical to any antibody sequences, especially notto any CDR sequences, presently known. The sequences themselves can bewholly novel ab initio and not be exactly represented in any antibodyproduced by any living organism. Thus, the high affinity CDR sequencesdisclosed herein are found, or achieved, by optimization, as disclosedherein, and then, once said high affinity CDR sequences are known, canbe used to synthesize fully functional antibody molecules, whetherdimeric or tetrameric, bifunctional or monofunctional, by any and allmeans known to science.

[0072] In keeping with the foregoing, and in order to better describethe sequences disclosed according to the invention, including theiroptimization, the sequence of the light and heavy chain variable regionsof a reference antibody (here, the anti-RSV antibody of U.S. Pat. No. 5,5,824,307) are shown in FIG. 1A (light chain variable region—SEQ IDNO: 1) and FIG. 1B (heavy chain variable region—SEQ ID NO: 2). Also inaccordance with the invention, novel sequences were produced with aminoacid differences only in CDR regions relative to the reference antibody.One means utilized to accomplish this result was to introduce mutationsin CDR regions of the so-called starting or reference chains and thenassay the resulting recombinatorial clones for antigen (RSV F protein)affinity.

[0073] In accordance with the forgoing, changes were made in first oneCDR sequence to optimize that sequence and determine the “critical”residue, or residues, than said position(s) was optimized through aseries of amino acid substitutions limited to that position alone. Eachof the 6 CDRs of the antibody clone was studied in turn until the“critical” CDRs were determined (wherein “critical CDRs” means CDRshaving a substantial effect on antibody binding, such as the beneficialor high affinity CDRs of Table 3). No all CDRs were found to becritical. For the antibody used in this particular study, only CDRs H1,H3, L2, and L3 were found to be critical but the results may bedifferent for a different antibody. Once a “high affinity” CDR wasdetermined (i.e., a “beneficial CDR”) then combinations of the CDRs werestudied to optimize the combination of CDR sequences resulting in a highaffinity neutralizing antibody of the invention.

[0074] As a very specific embodiment, the invention disclosed hereinrelates to a high affinity neutralizing antibody against respiratorysyncytial virus (RSV) having an affinity constant of at least 10¹⁰ M⁻¹,wherein said affinity constant could be within at least 2 fold of thisvalue because of the variability of such determinations and thevariability of affinity of the different cloned antibodies for theantigen (here, F antigen of RSV). Some of the resulting optimizedstructures within this embodiment had K_(a) greater than 10¹¹ M⁻¹ (forexample, clones numbered 22 and 23 in Table 4).

[0075] This high affinity neutralizing antibody is also an antibody thatbinds to the same epitope on RSV as the antibody whose light chainvariable region has the sequence of SEQ ID NO: 1 (FIG. 1A) and whoseheavy chain variable region has the sequence of SEQ ID NO: 2 (FIG. 1B).

[0076] In general, the approach used to identify antibodies of theinvention, based on the specific example of anti-RSV just described, wasto generate nucleotide sequences for the genes expressing the desiredantibody chains and insert these into vectors then used to transformEscherichia coil cells by standard protocols. The cells were grown inwells and the supernatant sampled and measured for antigen binding usingcapture lift and ELISA techniques. [See: Watkins et al, (1997) Anal.Biochem. 253, 37-45; Watkins et al, (1998) Anal. Biochem. 256, 169-177(the specifications of which are incorporated herein by reference).]These polynucleotides were designed so as to provide single amino acidreplacements in the CDRs that could then be screened for increasedaffinity, with beneficial replacements (those yielding increasedaffinity) being selectively combined for increased affinity. These werethen screened for binding affinity for F antigen of RSV versus the basicor reference antibody.

[0077] Using this protocol, ELISA data indicated that no single aminoacid replacements in CDRs L1 or H2 produced any increase in the affinityof the antibody clone for the epitope used as antigen (here, the Fantigen of RSV). Therefore, the antibodies of the present invention allcontain CDR sequences that differ from the reference antibody only inCDRs L2, L3, H1 and H3 (here, the reference antibody with sequences inFIG. 1 was merely a useful reference against which to test proceduresfor optimization of antibody affinity by increasing K_(a) and any othersystem could be used equally well).

[0078] The antibodies thus disclosed herein with respect to RSV alsocommonly have framework regions derived from a human antibody but, wherenot so derived, preferably from a mouse.

[0079] For the CDRs of the reference antibody, the amino acid sequenceof each CDR (as given in the sequences of FIG. 1) is shown in Table 1.Amino acid residue locations within the CDRs of the basic or referenceantibody, which, if replaced by amino acids as taught by the presentinvention, following optimization, produced high affinity CDR sequences(resulting in very high affinity neutralizing antibodies) and thereby abeneficial result (increase in affinity) are indicated in bold face andunderlining in Table 1 (such sequences giving increased affinity overthe reference antibody being denoted as “beneficial CDRs” or “highaffinity CDRs”). The CDRs of the basic or reference antibody (FIG. 1)are referred to herein as “basic or reference CDRs”). Thus, Table 1represents the CDR sequences depicted in FIG. 1 (i.e., for the anti-RSVreference antibody used herein to monitor optimization). TABLE 1Sequences of Basic or Reference CDRs CDR Length Sequence SEQ ID NO. L110 SASSSVGYMH 3 L2 7 DTSKLAS 4 L3 9 FQGSGYPFT 5 H1 7 TSGMSVG 6 H2 16DIWWDDKKDYNPSLKS 7 H3 10 SMITNWYFDV 8

[0080] With respect to the sequences disclosed herein, the CDR regionsas defined for purposes of the present invention are those segmentscorresponding to residues 24-33 (CDR L1), 49-55 (CDR L2) and 88-95 (CDRL3) of the light chain variable regions and residues 31-37 (CDR H1),52-67 (CDR H2) and 100-109 (CDR H3) of the heavy chain variable regionsof the antibodies disclosed herein.

[0081] In producing the antibodies of the present invention, whether bygenerating clones or cloning the polypeptide chains composing saidantibodies, or by direct synthesis of the polypeptide sequences, with orwithout the use of polynucleotide sequences coding therefor, or bywhatever method the user may choose, since no method of producing saidantibodies results in a limitation of the teaching of the presentinvention, the basic or reference antibody (heavy and light chainvariable regions (CDRs plus Framework) shown in FIG. 1) can be used as a“template” for generating the novel CDR sequences of the antibodies ofthe present invention and for purposes of comparing binding constants,etc. Standard approaches to characterizing and synthesizing the six CDRlibraries of single mutations were used (see Wu et al, Proc. Natl. Acad.Sci. 95:6037-6042 (1998)). The target CDR was first deleted for each ofthe libraries prior to annealing the nucleotides. For synthesis of thelibraries, the CDRs were defined as in Table 1. Codon based mutagenesisfor oligonucleotide synthesis to yield the CDR sequences of theinvention was employed (as described above).

[0082] Libraries were initially screened by capture lift to identify thehighest affinity variants. Subsequently, these clones were furthercharacterized using capture ELISA and by titration on immobilizedantigen.

[0083] DNA from the highest affinity variants was sequenced to determinethe nature of the beneficial or high affinity replacements. Afterscreening, it was determined that eight beneficial or high affinityreplacements, occurring in only four of the CDRs, had been observed.These are summarized as the CDR sequences in Table 3 with differencesversus the reference or basic CDRs of Table 1 being bold and underlined.Thus, the CDR sequences of Table 3 can be considered a CDR library ofcassettes available for use in producing a high affinity neutralizingantibody of the present invention where specificity is directed towardthe F antigen of RSV.

[0084] Analysis of the data indicated that replacement of amino acids atselected locations had greatly increased epitope binding, especiallywhere the nature of the replacement was the insertion of an amino acidselected from the group phenylalanine, alanine, proline, tryptophan andtyrosine, most especially phenylalanine, all such beneficial or highaffinity replacements again being at selected positions.

[0085] For the optimization experiment described herein and employingthe RSV/anti-RSV system, the most beneficial of high affinity CDRs werefound to result from amino acid replacements in 3 or 4 of the 6 CDRs,and in just 4 amino acid locations overall. Thus, the high affinityneutralizing antibodies of the present invention contain amino acidsequences differing from that of the base or reference antibody only incomplementarity determining regions, or in such regions as well as insurrounding framework regions, unlike previously known man-madeantibodies. Thus, the antibodies of the present invention are highaffinity neutralizing antibodies containing one or more CDR sequencesselected so as to produce high affinity in the antibody molecule. In thespecific embodiment using RSV as the target such differences are foundonly in L2 (or CDRL2), L3 (or CDRL3), H1 (or CDRH1) and H3 (or CDRH3).As noted, the greatest affinities for this antibody occurred only atselected amino acid positions of these CDRs, and in some of theembodiments of the present invention just one amino acid location ineach CDR was preferred for giving high affinity.

[0086] Thus, for CDR H1, substitution at amino acid 2 of the CDR(counting from the amino terminal end of the particular underlined CDRsequence of FIG. 1B), especially by replacing the serine located atposition 2 of CDR H1 of the basic or reference antibody with either analanine or a proline, was found to be most beneficial and therefore toresult in higher affinity for the RSV antigen epitope. For CDR H3,replacement of the glycine at position 6 of the CDR sequence, especiallyby either a phenylalanine or tryptophan, most especially byphenylalanine, was found to result in increased affinity for the RSVepitope. For CDR L2, replacement of the serine at position 3 of the CDR,especially by either a phenylalanine or a tyrosine, resulted inincreased affinity for F antigen. For CDR L3, replacement of the glycineat position 5 of the CDR, especially by phenylalanine, tryptophan ortyrosine, resulted in increased affinity for the RSV epitope.

[0087] In accordance with the invention, by combining such amino acidsubstitutions so that more than one occurred in the same antibodymolecule, it was possible to greatly increase the affinity of theantibodies disclosed herein for the epitope of the F antigen of RSV.

[0088] Table 2 shows the results of using the novel CDR sequences (forH1, H3, L2, and L3, respectively) of a number of clones according to thepresent invention. As shown in Table 2, a particular antibody may haveincorporated therein as many as 1, 2, or 3 novel CDRs of the inventionversus the basic or reference antibody chains shown in FIGS. 1A and 1B(for light (L2 and L3) and heavy (H1 and H3) chains, respectively). Theeffects of the novel CDRs are described in terms of Antigen (Ag)titration score (see below), where the basic or reference antibody isshown at the top and has a score of 0.1. Other scores are indicatedrelative to this 0.1 score of the “basic or reference antibody”sequences. The identities of the amino acids giving highest affinitiesat the respective locations (i.e., position 2 for H1, position 6 for H3,position 3 for L2, and position 5 for L3) are indicated below the aminoacids for the basic or reference antibody merely to indicate the rangeof mutations used for each CDR.

[0089] The total number of clones examined in this experiment was 37,with some duplicates (indicated by the “n” value in parenthesis, forexample, “n=4” for clone No. 7 indicates that 4 duplicate clones wereexamined).

[0090] In general, the data showed that there is a correlation betweenaffinity and the number of beneficial or high affinity CDRs, with all ofthe higher affinity variants having more than one beneficial or highaffinity CDR. Further, all of the best clones had an F (Phe) at position6 within CDR H3. Also, the beneficial or high affinity CDRs were thosewherein a hydrophobic, especially an aromatic, amino acid was insertedin place of the residue found in the basic or reference antibody.

[0091] The antibody titration assay employed varying concentrations ofantibody using 500 ng of RSV F antigen for each measurement. A graph ofcomparison data for a number of the combinatorial clones of Table 2 isshown in FIG. 2.

[0092] In sum, Table 2 shows a number of clones evaluated by theprocedures described herein along with the amino acids occurring at thekey locations (underlined and bold-faced in Tables 1 and 3) of CDRs H1,H3, L2, and L3. The Table also summarizes the number of differencesbetween the CDRs of these clones versus the corresponding CDRs of thereference antibody (See Table 1 and FIG. 1). The right side of the Tableshows an “Ag Score” or antigen binding value, which represents anarbitrary and qualitative value, ranging from 0-4, and represents aqualitative estimate of the relative binding ability of the differentantibody clones based on their respective titration curves. This valueis provided here only for rough qualitative comparisons of the differentantibodies and is not intended as a quantitative measure of bindingability. TABLE 2 Summary of Clone Data CDRs Clone H1 H3 L2 L3 # NovelCDRs Ag Score Basic S W S G 0 0.1 Single A F F F P Y W Y  1 A F S F 3 4 2 A F F G 3 4  3 (n = 3) P F F F 4 4  4 (n = 3) P F F Y 4 3.5  5 (n =3) P F F W 4 3.5  6 P F Y F 4 3.5  7 (n = 4) P F F G 3 3  8 P F F ?  3+3.5  9 (n = 2) P F S W 3 3 10 P F S F 3 3 11 P W F W 3 3 12 (n = 2) P WF F 3 2.5 13 (n = 3) S F F F 3 2.5 14 S F F W 3 2.5 15 (n = 2) A F S G 22.5 16 (n = 2) P F S G 2 2 17 P W S W 2 2 18 (n = 2) S F F G 2 2 19 S FS W 2 2 20 S F S F 2 2 21 S W Y F 2 2

[0093] Table 2 also shows the number of novel CDRs for each antibodyclone (meaning the number of CDRs in the antibody with at least oneamino acid difference with respect to the corresponding CDR of thereference antibody—see Table 1). The number of novel CDRs is also thenumber of “beneficial” or “high affinity” CDRs present in that antibodymolecule. The amino acid differences in the novel CDR would occur at theposition bold and underlined in Table 1 for the reference antibody sothat the amino acid bold and underlined in Table 1 has been replaced bythe amino acid indicated for the respective CDR in Table 2 (usingstandard single letter amino acid designations).

[0094] The novel CDRs represented in each of the clones is readilydetermined by locating the clone in the table, and matching theindicated amino acid for each CDR with the corresponding amino acid forthe same CDR next to the basic or reference clone. For convenience,where an amino acid is different in a particular CDR of one of theclones, the new amino acid is indicated in bold face. In addition, forall clones shown in the table, replacements occur only at the selectedlocations recited above as yielding a novel CDR. Thus, all substitutionsin CDR H1 relative to the basic or reference antibody are at position 2of the CDR (meaning, again, the second amino acid from the N-terminalend of CDR H1 as underlined and bold-faced in FIG. 1 and Tables 1 and3), all substitutions in CDR H3 are at position 6, all substitutions inCDR L2 are at position 3, and all substitutions in CDR L3 are atpositions 5, again all with reference to the basic or referenceantibody. It should be kept in mind that the basic or reference antibodywas chosen because it was known already to have a very high affinity forRSV-epitopes. [See: Johnson et al, (1997) J. Infect. Dis., 176,1215-1224] So, for example, the table shows that for clone No. 1, forthe beneficial or high affinity CDR H1, an alanine is used in place ofthe serine at position 2 of CDR H1 of the basic or reference antibody,thereby achieving an increased affinity for RSV, and a phenylalanineoccurs in place of the tryptophan at position 6 of CDR H3, the serine atposition 3 of CDR L2 of the basic or reference antibody was used and aphenylalanine occurred at position 5 of beneficial or high affinity CDRL3.

[0095] Thus, the novel and beneficial CDRs according to the presentinvention (i.e., high affinity CDRs or CDR sequences whose presence inthe basic or reference antibody in place of the corresponding basic orreference CDR served to greatly increase the affinity of said antibodyfor the same RSV epitope) which are present in the antibody structuresproduced in the supernatants tested for the clones of Table 2 aresummarized in Table 3. In each case, the bold face indicates how thenovel and beneficial, or high affinity, CDRs of the invention differfrom the corresponding CDRs of the basic or reference anti-RSV antibody.TABLE 3 Sequences for High Affinity CDRs CDR Sequence SEQ ID NO: H1TAGMSVG 9 H1 TPGMSVG 10 H3 SMITNFYFDV 11 L2 DTFKLAS 12 L2 DTYKLAS 13 L3FQGSFYPFT 14 L3 FQGSYYPFT 15 L3 FQGSWYPFT 16

[0096] While the CDR sequences of Table 3 represent the sequences forthe high affinity CDRs disclosed according to the invention, it isunderstood that one or more of these CDRs may be present in the sameantibody and the sequences of the table indicate the set from whichappropriate sequences for each of the high affinity CDRs may beselected. Thus, as shown in Table 3, when a high affinity H1 CDR ispresent in a high affinity neutralizing antibody of the inventiondisclosed herein, it has a sequence corresponding to the sequence of SEQID NO: 9 or 10. When a neutralizing antibody of the claimed inventioncontains a high affinity H3 CDR, said CDR has the sequence of SEQ ID NO:12. When a high affinity neutralizing antibody of the invention containsa high affinity L2 CDR, said high affinity L2 CDR has an amino acidsequence selected from the group consisting of the sequences of SEQ IDNO: 12 and 13. Finally, when a high affinity neutralizing antibody ofthe present invention contains a high affinity L3 CDR, said CDR has anamino acid sequence selected from the group consisting of the sequencesof SEQ ID NO: 14, 15 and 16.

[0097] As already stated, in one embodiment, the high affinityneutralizing antibodies are antibodies that include a human constantregion.

[0098] Thus, in a preferred embodiment, the high affinity neutralizingantibody of the invention, with an affinity of at least 10¹⁰ M⁻¹, oreven at least 10¹¹ M⁻¹, is a grafted antibody that includes a humanconstant region and a framework for the heavy and light chains whereinat least a portion of the framework is derived from a human antibody (orfrom a consensus sequence of a human antibody framework).

[0099] In another embodiment, all of the framework is derived from ahuman antibody (or a human consensus sequence).

[0100] Thus, an RSV-neutralizing antibody, with an affinity of at least10¹⁰ M⁻¹, is a grafted antibody having a human constant region, one ormore CDRs that are derived from a non-human antibody in which at leastone of the amino acids in at least one of said CDRs is changed and inwhich all or a portion of the framework is derived from a human antibody(or a consensus sequence of a human antibody framework).

[0101] Because the combination of CDR sequences of one antibody withnon-CDR regions of another antibody results from a form of “grafting” ofCDRs onto the remainder of the molecule, these have been referred to as“CDR grafted” antibodies. Today, using the techniques of geneticengineering the same product can be formed without isolating anysequences from actual antibodies. So long as the desired CDR sequences,and the constant and framework sequence are known, genes with thedesired sequences can be assembled and, using a variety of vectors,inserted into appropriate cells for expression of the functionaltetrameric antibody molecules. Coupling this with the methodologyalready described permits the assembly of single mutation librarieswherein the antibodies possess the same sequences as correspondinggrafted antibodies and, therefore, the same structure and bindingaffinities.

[0102] The high affinity antibodies of the invention can be present in arelatively pure or isolated form as well as in a supernatant drawn fromcells grown in wells or on plates. Such supernatants were used togenerate the data of Table 1. The antibodies of the invention can thusalso be present in the form of a composition comprising the antibody ofthe invention and wherein said antibody is suspended in apharmacologically acceptable carrier, or excipient. The antibodies ofthe invention may be present in such a composition at a concentration,or in an amount, sufficient to be of therapeutic or pharmacologicalvalue in treating diseases, such as RSV. Said antibodies may also bepresent in a composition in a more dilute form.

[0103] Consequently, the invention is also directed to providing amethod of preventing and/or treating respiratory syncytial virusinfections comprising the administering to a patient at risk thereof, orafflicted therewith, of a therapeutically (including prophylactically)effective amount of the antibody composition described herein.

[0104] One preferred embodiment of the high affinity antibodies of thepresent invention is the antibody whose heavy and light chain CDRregions have sequences as follows: CDR H1 has the sequence of SEQ ID NO:9, CDR H3 has the sequence of SEQ ID NO: 11, CDR L2 has the sequence ofSEQ ID NO: 4 (no change from CDR L2 of the reference sequence of FIG.1A), and CDR L3 has the sequence of SEQ ID NO: 14 (see Table 3). In thispreferred embodiment, the affinity constant is about 6.99×10¹⁰ (or about14.3 pM as a dissociation constant) as shown in Table 4 (clone 1). Theheavy and light chain variable regions of an antibody comprising thisembodiment, along with framework sequences, is shown in FIG. 3.

[0105] Another preferred embodiment of the high affinity antibodies ofthe present invention is the antibody whose heavy and light chain CDRregions have the sequences as follows: CDR H1 has the sequence of SEQ IDNO: 9, CDR H3 has the sequence of SEQ ID NO: 11, CDR L2 has the sequenceof SEQ ID NO: 12, and CDR L3 has the sequence of SEQ ID NO: 5 (see Table2, clone 2—no difference from the reference sequence of CDR L3 of FIG.1A). In this preferred embodiment, the affinity constant is about7.30×10¹⁰ (or about 13.7 pM as a dissociation constant) as shown inTable 4 (clone 2).The heavy and light chain variable regions of anantibody comprising this embodiment, along with framework sequences, isshown in FIG. 4.

[0106] An additional preferred embodiment of the high affinityantibodies of the present invention is the antibody whose heavy andlight chain CDR regions have the sequences as follows: CDR H1 has thesequence of SEQ ID NO: 10, CDR H3 has the sequence of SEQ ID NO: 11, CDRL2 has the sequence of SEQ ID NO: 12, and CDR L3 has the sequence of SEQID NO: 14 (see Table 3 for CDR sequences) which clone is designatednumber 3 in Table 2. In this preferred embodiment, the affinity constantis about 8.13×10¹⁰ (or about 12.3 pM as a dissociation constant) asshown in Table 4 (clone 3). The heavy and light chain variable regionsof an antibody comprising this embodiment, along with frameworksequences, is shown in FIG. 5.

[0107] A most preferred embodiment of the high affinity antibodies ofthe present invention is the antibody whose heavy and light chain CDRregions have the sequences as follows: CDR Hi has the sequence of SEQ IDNO: 9, CDR H3 has the sequence of SEQ ID NO: 11, CDR L2 has the sequenceof SEQ ID NO: 12, and CDR L3 has the sequence of SEQ ID NO: 14 (seeTable 3). In this preferred embodiment, the affinity constant is about3.6×10¹¹ (or about 2.8 pM as a dissociation constant) as shown in Table4 (clone 22). The heavy and light chain variable regions of an antibodycomprising this embodiment, along with framework sequences, is shown inFIG. 6.

[0108] Another most preferred embodiment of the present invention is theantibody whose heavy and light chain CDR regions include the followingsequences: CDR H1 has the sequence of SEQ ID NO: 9, CDR H3 has thesequence of SEQ ID NO: 11, CDR L2 has the sequence of SEQ ID NO: 12, andCDR L3 has the sequence of SEQ ID NO: 15 (see Table 3). In this latterpreferred embodiment, the affinity constant is about 4×10¹¹ (about 2.5pM as a dissociation constant) as shown in Table 4 (clone 23). The heavyand light chain variable regions of an antibody comprising thisembodiment, along with framework sequences, is shown in FIG. 7.

[0109] In particularly preferred embodiments, the antibodies of thepresent invention will have the framework regions of the sequencesdepicted for the framework regions shown in FIGS. 1, 3, 4, 5, 6, and 7(each contains the same framework regions and differ only in CDRsequences). These most preferred embodiments include the neutralizingantibody wherein the light chain variable region has the amino acidsequence of SEQ ID NO: 17 and the heavy chain variable region has theamino acid sequence of SEQ ID NO: 18; the neutralizing antibody whereinthe light chain variable region has the amino acid sequence of SEQ IDNO: 19 and the heavy chain variable region has the amino acid sequenceof SEQ ID NO: 20; the neutralizing antibody wherein the light chainvariable region has the amino acid sequence of SEQ ID NO: 21 and theheavy chain variable region has the amino acid sequence of SEQ ID NO:22; the neutralizing antibody wherein the light chain variable regionhas the amino acid sequence of SEQ ID NO: 23 and the heavy chainvariable region has the amino acid sequence of SEQ ID NO: 24; theneutralizing antibody wherein the light chain variable region has theamino acid sequence of SEQ ID NO: 25 and the heavy chain variable regionhas the amino acid sequence of SEQ ID NO: 26.

[0110] It should be kept in mind that while the high affinityneutralizing antibodies of the present invention can be assembled fromCDR regions and non-CDR regions derived from actual neutralizingantibodies by splicing amino acid segments together (and antibodies soassembled would be within the invention disclosed herein) the antibodiesof the present invention are most conveniently prepared by geneticallyengineering appropriate gene sequences into vectors that may then betransfected into suitable cell lines for eventual expression of theassembled antibody molecules by the engineered cells. In fact, suchrecombinant procedures were employed to prepare the antibodies disclosedherein. In addition, because the sequences of the chains of the highaffinity antibodies are known from the disclosure herein, suchantibodies could also be assembled by direct synthesis of theappropriate chains and then allowed to self-assemble into tetrameric(H₂L₂) bivalent antibody structures.

[0111] The method of preparing the high affinity neutralizing antibodiesof the invention involved the creation of a combinatorial library whichwas used to prepare clones producing antibodies comprising thebeneficial CDRs of the invention that could then be screened foraffinity for RSV epitopes (for Example, FIG. 2).

EXAMPLE 1 Kinetic Analysis of Humanized RSV Mabs by BIAcorekTM

[0112] The kinetics of interaction between high affinity anti-RSV Mabsand the RSV F protein was studied by surface plasmon resonance using aPharmacia BlAcoreTM biosensor. A recombinant baculovirus expressing aC-terminal truncated F protein provided an abundant source of antigenfor kinetic studies. The supernatant, which contained the secreted Fprotein, was enriched approximately 20-fold by successive chromatographyon concanavalin A and Q-sepharose columns. The pooled fractions weredialyzed against 10 mM sodium citrate (pH 5.5), and concentrated toapproximately 0.1 mg/ml. In a typical experiment, an aliquot of theF-protein (100 ml) was amine-coupled to the BIAcore sensor chip. Theamount immobilized gave approximately 2000 response units (R_(max)) ofsignal when saturated with the mouse monoclonal antibodies H1129 orH1308F (prepared as in U.S. Pat. 5,824,307, whose disclosure is herebyincorporated by reference). This indicated that there was an equalnumber of “A” and “C” antigenic sites on the F-protein preparationfollowing the coupling procedure. Two unrelated irrelevant Mabs (RVFV4D4 and CMV H758) showed no interaction with the immobilized F protein.A typical kinetic study involved the injection of 35 ml of Mab atvarying concentrations (25-300 nM) in PBS buffer containing 0.05%Tween-20 (PBS/Tween). The flow rate was maintained at 5 ml/min, giving a7 min binding phase. Following the injection of Mab, the flow wasexchanged with PBS/Tween buffer for 30 min for determining the rate ofdissociation. The sensor chip was regenerated between cycles with a 2min pulse of 10 mM HCl. The regeneration step caused a minimal loss ofbinding capacity of the immobilized F-protein (4% loss per cycle). Thissmall decrease did not change the calculated values of the rateconstants for binding and dissociation (also called the k_(on) andk_(off), respectively).

[0113] More specifically, for measurement of k_(assoc) (or k_(on)), Fprotein was directly immobilized by the EDC/NHS method(EDC=N-ethyl-N′-[3-diethylamino-propyl]carbodiimide;NHS=N-hydroxysuccinimide) with the F-protein being injected over theEDC/NHS activated sensor chip. Briefly, 4 μg/ml of F protein in 10 mMNaOAc, pH 4.0 was prepared and about a 30 μl injection gives about 500RU (response units) of immobilized F protein under the above referencedconditions. The blank flow cell (VnR immobilized-CM dextran surface) wassubtracted for kinetic analysis. The column could be regenerated using100 mM HCl (with 72 seconds of contact time being required for fullregeneration). This treatment removed bound Fab completely withoutdamaging the immobilized antigen and could be used for over 40regenerations. For k_(on) measurements, Fab concentrations were 12.5 nM,25 nM, 50 nM, 100 nM, 200 nM, and 400 nM. The dissociation phase wasanalyzed from 230 seconds (30 seconds after start of the dissociationphase) to 900 seconds. Kinetics were analyzed by 1:1 Langmuir fitting(global fitting). Measurements were done in HBS-EP buffer (10 mM HEPES,pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.005% (v/v) Surfactant P20.

[0114] For measurements of combinatorial clones, as disclosed herein,the k_(on) and k_(off) were measured separately. The k_(on) was measuredat conditions that were the same as those for the single mutation clonesand was analyzed similarly.

[0115] For measuring k_(dissoc) (or k_(off)), the following conditionswere employed. Briefly, 4100 RU of F protein were immobilized (as above)with CM-dextran used as the blank. Here, 3000 RU of Fab was bound (withdissociated Fab high enough to offset machine fluctuation). HBS plus 5nM F protein (about 350-2000 times higher than the K_(dissoc) orK_(d)—the dissociation equilibrium constant) was used as buffer. Thedissociation phase was 6-15 hours at a flow rate of 5 μl/min. Under theconditions used herein, re-binding of the dissociated Fab was minimal.For further details, see the manual with the biosensor.

[0116] The binding of the high affinity anti-RSV antibodies to the Fprotein, or other epitopic sites on RSV, disclosed herein was calculatedfrom the ratio of the first order rate constant for dissociation to thesecond order rate constant for binding or association(K_(d)=k_(diss)/k_(assoc)). The value for kassoc was calculated based onthe following rate equation:

dR/dt=k _(assoc) [Mab]R _(max)−(k _(assoc) [Mab]+k _(diss))R

[0117] where R and R_(max) are the response units at time t andinfinity, respectively. A plot of dr/dt as a function of R gives a slopeof (k_(assoc)[Mab]+kd_(diss))—Since these slopes are linearly related tothe [Mab], the value k_(assoc) can be derived from a replot of theslopes versus [Mab]. The slope of the new line is equal to k_(assoc).Although the value of k_(diss) can be extrapolated from the Y-intercept,a more accurate value was determined by direct measurement of k_(diss).Following the injection phase of the Mab, PBS/Tween buffer flows acrossthe sensor chip. From this point, [Mab]=0. The above stated equation fordR/dt thus reduces to:

dr/dt=k

[0118] or

dR/R=k _(diss) dt

[0119] Integration of this equation then gives:

In(R ₀ /R _(t))=k _(diss) t

[0120] where R₀/R_(t)) are the response units at time 0 (start ofdissociation phase) and t, respectively. Lastly, plotting ln(R₀/R_(t))as a function of t gives a slope of k_(diss).

[0121] In the preferred embodiment herein, the numerical values fromsuch antibody variants were as follows: TABLE 4 Summary of KineticConstants from Ultra-high Affinity Antibodies. Clone ID CDRs k_(assoc)(M⁻¹sec⁻¹) k_(diss) (sec⁻¹) K_(a) (M⁻¹) 1 AFSF 1.13 × 10⁵ 1.62 × 10⁻⁶6.98 × 10¹⁰ 2 AFFG 1.33 × 10⁵ 1.82 × 10⁻⁶ 7.31 × 10¹⁰ 3 PFFF 1.10 × 10⁵1.35 × 10⁻⁶ 8.15 × 10¹⁰ 22 AFFF 1.34 × 10⁵ 3.70 × 10⁻⁷ 3.62 × 10¹¹ 23AFFY 1.22 × 10⁵ 3.03 × 10⁻⁷ 4.03 × 10¹¹

[0122] Here, the CDRs represent the amino acids replacing the referenceamino acids at the key positions (or critical positions) of the CDRsshown in Table 1 (in bold and underlined) for a reference antibody.Thus, for example, clone 22 has an alanine at position 2 of CDR H1(residue 32 of the heavy chain variable region —SEQ ID NO: 24) in placeof the serine shown at that position in Table 1 (SEQ ID NO: 6), aphenylalanine at position 6 of CDR H3 (residue 105 of the heavy chainvariable region—SEQ ID NO: 24) in place of the tryptophan shown at thatposition in Table 1 (SEQ ID NO: 8), a phenylalanine at position 3 of CDRL2 (residue 51 of the light chain variable region—SEQ ID NO: 23) inplace of the serine shown at that position in Table 1 (SEQ ID NO: 4),and a phenylalanine at position 5 of CDR L3 (residue 92 of the lightchain variable region—SEQ ID NO: 23) in place of the glycine shown atthat position in Table 1 (SEQ ID NO: 5).

[0123] Of course, in forming such clones, Table 3 represents a pool ofpotential CDRs from which the high affinity CDRs of the antibodies ofthe present invention are to be drawn. For example, clone 23 of Table 4uses the same CDRs as clone 22 with the exception of CDR L3, which has atyrosine at position 5 of CDR L3 (Table 3—SEQ ID NO: 15) in place of theglycine shown in that position in Table 1 (SEQ ID NO: 5). Thesubstitutions at the corresponding critical positions are likewise shownin Table 2.

EXAMPLE 2 Microneutralization Assay

[0124] Neutralization of the antibodies of the present invention weredetermined by microneutralization assay. This microneutralization assayis a modification of the procedures described by Anderson et al (1985).Antibody dilutions were made in triplicate using a 96-well plate. TenTCID₅₀ of respiratory syncytial virus (RSV—Long strain) were incubatedwith serial dilutions of the antibody (or Fabs) to be tested for 2 hoursat 37° C. in the wells of a 96-well plate. RSV susceptible HEp-2 cells(2.5×10⁴) were then added to each well and cultured for 5 days at 37° C.in 5% CO₂. After 5 days, the medium was aspirated and cells were washedand fixed to the plates with 80% methanol and 20% PBS. RSV replicationwas then determined by F protein expression. Fixed cells were incubatedwith a biotin-conjugated anti-F protein monoclonal antibody (pan Fprotein, C-site-specific MAb 133-1H) washed and horseradish peroxidaseconjugated avidin was added to the wells. The wells were washed againand turnover of substrate TMB (thionitrobenzoic acid) was measured at450 nm. The neutralizing titer was expressed as the antibodyconcentration that caused at least 50% reduction in absorbency at 450 nm(the OD₄₅₀) from virus-only control cells. Results for severalantibodies of the present invention are shown by the graph in FIG. 8while results using Fab fragments are depicted in the graph of FIG. 9.

BACKGROUND AND CITED REFERENCES

[0125] 1. Hall, C. B., Douglas, R. G., Geiman, J. M. et al.,N.Engl.J.Med. 293:1343, 1975.

[0126] 2. Hall, C. B., McBride, J. T., Walsh, E. E. et al.,N.Engl.J.Med. 308:1443, 1983.

[0127] 3. Hall, C. B., McBride, J. T., Gala, C. L. et al., JAMA254:3047,1985.

[0128] 4. Wald, E. R., et al., J.Pediat. 112:154,1988.

[0129] 5. Kapikian, A. Z., Mithcell, R. H., Chanock, R. M. et al.,Am.J.Epidemiol. 89:405,1969.

[0130] 6. Prince, G. A., Hemming, V. G., Horswood, R. L. et al., VirusRes. 3:193, 1985.

[0131] 7. Hemming, V. G., Prince, G. A., Horswood, R. L. et al.,J.Infect.Dis. 152:1083,1985.

[0132] 8. Wright, P. F., Belshe, R. B., et al., lnfect.lmmun.37:397,1982.

[0133] 9. Conrad, D. A., Christenson, J. C., et al.,Peditr.lnfect.Dis.J. 6:152,1987.

[0134] 10. LoBuglio, A. F., Wheeler, R. L., Trang, J. et al.,Proc.Natl.Acad. Sci. 86:4220,1989.

[0135] 11. Steplewski, Z., Sun, L. K., Shearman, C. W. et al.,Proc.Natl.Acad. Sci. 85:4852,1988.

[0136] 12. Boulianne, G. L., Hozumi, N., Shulman, M. J. Nature.312:643,1984.

[0137] 13. Sun, L. K., Curtis, P., Rakowicz-Szulczynska, E. et al.,Proc.Natl.Acad. Sci. 84:214, 1987.

[0138] 14. Liu, A. Y., Mack, P. W., Champion, C. I., Robinson, R. R.,Gene 54:33,1987.

[0139] 15. Morrison, S. L., Johnson, M. J., Hersenber, L. A., Oi, V. T.Proc.Natl.Acad. Sci. 81:6851,1984.

[0140] 16. Morrison, S. L. Science 229:1202, 1985.

[0141] 17. Sahagan, B. G., Dorai, H., Saltzgaber-Muller, J. et al.,J.Immunol. 137:1066, 1986.

[0142] 18. Taked, S., Naito, T., Hama, K., Noma, T., Honjo, T., Nature314:452,1985.

[0143] 19. Carson, D. A., Freimark, B. D., Adv. Immunol. 38:275,1986.

[0144] 20. Beeler, J. A., et al., J.Virol. 63:2941-2950,1989.

[0145] 21. Coelingh, et al., Virology, 143:569-582,1985.

[0146] 22. Anderson et al. Microneutralization test for respiratorysyncytial virus based o an enzyme immunoassay, J. Clin. Microbiol.(1985) 22:1050-1052.

[0147] 23. Johnson et al., Development of a humanized monoclonalantibody (MEDI-493) with potent in vitro and in vivo activity againstrespiratory syncytial virus, J. Infectious Diseases (1997) 176:1215-1224.

1 26 1 106 PRT Artificial Sequence Description of ArtificialSequenceMouse human chimeric antibody light chain variable chain 1 AspIle Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly 1 5 10 15Asp Arg Val Thr Ile Thr Cys Ser Ala Ser Ser Ser Val Gly Tyr Met 20 25 30His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr 35 40 45Asp Thr Ser Lys Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser 50 55 60Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Asp 65 70 7580 Asp Phe Ala Thr Tyr Tyr Cys Phe Gln Gly Ser Gly Tyr Pro Phe Thr 85 9095 Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 2 120 PRT ArtificialSequence Description of Artificial SequenceMouse human chimeric antibodyheavy chain variable chain 2 Gln Val Thr Leu Arg Glu Ser Gly Pro Ala LeuVal Lys Pro Thr Gln 1 5 10 15 Thr Leu Thr Leu Thr Cys Thr Phe Ser GlyPhe Ser Leu Ser Thr Ser 20 25 30 Gly Met Ser Val Gly Trp Ile Arg Gln ProPro Gly Lys Ala Leu Glu 35 40 45 Trp Leu Ala Asp Ile Trp Trp Asp Asp LysLys Asp Tyr Asn Pro Ser 50 55 60 Leu Lys Ser Arg Leu Thr Ile Ser Lys AspThr Ser Lys Asn Gln Val 65 70 75 80 Val Leu Lys Val Thr Asn Met Asp ProAla Asp Thr Ala Thr Tyr Tyr 85 90 95 Cys Ala Arg Ser Met Ile Thr Asn TrpTyr Phe Asp Val Trp Gly Gln 100 105 110 Gly Thr Thr Val Thr Val Ser Ser115 120 3 10 PRT Artificial Sequence Description of ArtificialSequenceAmino acid sequence of complementarity determining region L1 ofreference anti-RSV antibody 3 Ser Ala Ser Ser Ser Val Gly Tyr Met His 15 10 4 7 PRT Artificial Sequence Description of Artificial SequenceAminoacid sequence of complementarity determining region L2 of referenceanti-RSV antibody 4 Asp Thr Ser Lys Leu Ala Ser 1 5 5 9 PRT ArtificialSequence Description of Artificial SequenceAmino acid sequence ofcomplementarity determining region L3 of reference anti-RSV antibody 5Phe Gln Gly Ser Gly Tyr Pro Phe Thr 1 5 6 7 PRT Artificial SequenceDescription of Artificial SequenceAmino acid sequence of complementaritydetermining region H1 of reference anti-RSV antibody 6 Thr Ser Gly MetSer Val Gly 1 5 7 16 PRT Artificial Sequence Description of ArtificialSequenceAmino acid sequence of complementarity determining region H2 ofreference anti-RSV antibody 7 Asp Ile Trp Trp Asp Asp Lys Lys Asp TyrAsn Pro Ser Leu Lys Ser 1 5 10 15 8 10 PRT Artificial SequenceDescription of Artificial SequenceAmino acid sequence of complementaritydetermining region H3 of reference anti-RSV antibody 8 Ser Met Ile ThrAsn Trp Tyr Phe Asp Val 1 5 10 9 7 PRT Artificial Sequence Descriptionof Artificial SequenceAmino acid sequence present in high affinitycomplementarity determining regions of antibodies of the invention 9 ThrAla Gly Met Ser Val Gly 1 5 10 7 PRT Artificial Sequence Description ofArtificial SequenceAmino acid sequence present in high affinitycomplementarity determining regions of antibodies of the invention 10Thr Pro Gly Met Ser Val Gly 1 5 11 10 PRT Artificial SequenceDescription of Artificial SequenceAmino acid sequence present in highaffinity complementarity determining regions of antibodies of theinvention 11 Ser Met Ile Thr Asn Phe Tyr Phe Asp Val 1 5 10 12 7 PRTArtificial Sequence Description of Artificial SequenceAmino acidsequence present in high affinity complementarity determining regions ofantibodies of the invention 12 Asp Thr Phe Lys Leu Ala Ser 1 5 13 7 PRTArtificial Sequence Description of Artificial SequenceAmino acidsequence present in high affinity complementarity determining regions ofantibodies of the invention 13 Asp Thr Tyr Lys Leu Ala Ser 1 5 14 9 PRTArtificial Sequence Description of Artificial SequenceAmino acidsequence present in high affinity complementarity determining regions ofantibodies of the invention 14 Phe Gln Gly Ser Phe Tyr Pro Phe Thr 1 515 9 PRT Artificial Sequence Description of Artificial SequenceAminoacid sequence present in high affinity complementarity determiningregions of antibodies of the invention 15 Phe Gln Gly Ser Tyr Tyr ProPhe Thr 1 5 16 9 PRT Artificial Sequence Description of ArtificialSequenceAmino acid sequence present in high affinity complementaritydetermining regions of antibodies of the invention 16 Phe Gln Gly SerTrp Tyr Pro Phe Thr 1 5 17 106 PRT Artificial Sequence Description ofArtificial Sequence Amino acid sequence of light chain variable regionof clone 1 of Figure 3A 17 Asp Ile Gln Met Thr Gln Ser Pro Ser Thr LeuSer Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Ser Ala SerSer Ser Val Gly Tyr Met 20 25 30 His Trp Tyr Gln Gln Lys Pro Gly Lys AlaPro Lys Leu Leu Ile Tyr 35 40 45 Asp Thr Ser Lys Leu Ala Ser Gly Val ProSer Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Glu Phe Thr Leu Thr IleSer Ser Leu Gln Pro Asp 65 70 75 80 Asp Phe Ala Thr Tyr Tyr Cys Phe GlnGly Ser Phe Tyr Pro Phe Thr 85 90 95 Phe Gly Gly Gly Thr Lys Val Glu IleLys 100 105 18 120 PRT Artificial Sequence Description of ArtificialSequenceAmino acid sequence of heavy chain variable region of clone 1 ofFigure 3B 18 Gln Val Thr Leu Arg Glu Ser Gly Pro Ala Leu Val Lys Pro ThrGln 1 5 10 15 Thr Leu Thr Leu Thr Cys Thr Phe Ser Gly Phe Ser Leu SerThr Ala 20 25 30 Gly Met Ser Val Gly Trp Ile Arg Gln Pro Pro Gly Lys AlaLeu Glu 35 40 45 Trp Leu Ala Asp Ile Trp Trp Asp Asp Lys Lys Asp Tyr AsnPro Ser 50 55 60 Leu Lys Ser Arg Leu Thr Ile Ser Lys Asp Thr Ser Lys AsnGln Val 65 70 75 80 Val Leu Lys Val Thr Asn Met Asp Pro Ala Asp Thr AlaThr Tyr Tyr 85 90 95 Cys Ala Arg Ser Met Ile Thr Asn Phe Tyr Phe Asp ValTrp Gly Gln 100 105 110 Gly Thr Thr Val Thr Val Ser Ser 115 120 19 106PRT Artificial Sequence Description of Artificial SequenceAmino acidsequence of light chain variable region of clone 2 of Figure 4A 19 AspIle Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly 1 5 10 15Asp Arg Val Thr Ile Thr Cys Ser Ala Ser Ser Ser Val Gly Tyr Met 20 25 30His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr 35 40 45Asp Thr Phe Lys Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser 50 55 60Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Asp 65 70 7580 Asp Phe Ala Thr Tyr Tyr Cys Phe Gln Gly Ser Gly Tyr Pro Phe Thr 85 9095 Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 20 120 PRT ArtificialSequence Description of Artificial SequenceAmino acid sequence of heavychain variable region of clone 2 of Figure 4B 20 Gln Val Thr Leu Arg GluSer Gly Pro Ala Leu Val Lys Pro Thr Gln 1 5 10 15 Thr Leu Thr Leu ThrCys Thr Phe Ser Gly Phe Ser Leu Ser Thr Ala 20 25 30 Gly Met Ser Val GlyTrp Ile Arg Gln Pro Pro Gly Lys Ala Leu Glu 35 40 45 Trp Leu Ala Asp IleTrp Trp Asp Asp Lys Lys Asp Tyr Asn Pro Ser 50 55 60 Leu Lys Ser Arg LeuThr Ile Ser Lys Asp Thr Ser Lys Asn Gln Val 65 70 75 80 Val Leu Lys ValThr Asn Met Asp Pro Ala Asp Thr Ala Thr Tyr Tyr 85 90 95 Cys Ala Arg SerMet Ile Thr Asn Phe Tyr Phe Asp Val Trp Gly Gln 100 105 110 Gly Thr ThrVal Thr Val Ser Ser 115 120 21 106 PRT Artificial Sequence Descriptionof Artificial SequenceAmino acid sequence of light chain variable regionof clone 3 of Figure 5A 21 Asp Ile Gln Met Thr Gln Ser Pro Ser Thr LeuSer Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Ser Ala SerSer Ser Val Gly Tyr Met 20 25 30 His Trp Tyr Gln Gln Lys Pro Gly Lys AlaPro Lys Leu Leu Ile Tyr 35 40 45 Asp Thr Phe Lys Leu Ala Ser Gly Val ProSer Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Glu Phe Thr Leu Thr IleSer Ser Leu Gln Pro Asp 65 70 75 80 Asp Phe Ala Thr Tyr Tyr Cys Phe GlnGly Ser Phe Tyr Pro Phe Thr 85 90 95 Phe Gly Gly Gly Thr Lys Val Glu IleLys 100 105 22 120 PRT Artificial Sequence Description of ArtificialSequenceAmino acid sequence of heavy chain variable region of clone 3 ofFigure 5B 22 Gln Val Thr Leu Arg Glu Ser Gly Pro Ala Leu Val Lys Pro ThrGln 1 5 10 15 Thr Leu Thr Leu Thr Cys Thr Phe Ser Gly Phe Ser Leu SerThr Pro 20 25 30 Gly Met Ser Val Gly Trp Ile Arg Gln Pro Pro Gly Lys AlaLeu Glu 35 40 45 Trp Leu Ala Asp Ile Trp Trp Asp Asp Lys Lys Asp Tyr AsnPro Ser 50 55 60 Leu Lys Ser Arg Leu Thr Ile Ser Lys Asp Thr Ser Lys AsnGln Val 65 70 75 80 Val Leu Lys Val Thr Asn Met Asp Pro Ala Asp Thr AlaThr Tyr Tyr 85 90 95 Cys Ala Arg Ser Met Ile Thr Asn Phe Tyr Phe Asp ValTrp Gly Gln 100 105 110 Gly Thr Thr Val Thr Val Ser Ser 115 120 23 106PRT Artificial Sequence Description of Artificial SequenceAmino acidsequence of light chain variable region of clone 22 of Figure 6A 23 AspIle Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly 1 5 10 15Asp Arg Val Thr Ile Thr Cys Ser Ala Ser Ser Ser Val Gly Tyr Met 20 25 30His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr 35 40 45Asp Thr Phe Lys Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser 50 55 60Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Asp 65 70 7580 Asp Phe Ala Thr Tyr Tyr Cys Phe Gln Gly Ser Phe Tyr Pro Phe Thr 85 9095 Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 24 120 PRT ArtificialSequence Description of Artificial SequenceAmino acid sequence of heavychain variable region of clone 22 of Figure 6B 24 Gln Val Thr Leu ArgGlu Ser Gly Pro Ala Leu Val Lys Pro Thr Gln 1 5 10 15 Thr Leu Thr LeuThr Cys Thr Phe Ser Gly Phe Ser Leu Ser Thr Ala 20 25 30 Gly Met Ser ValGly Trp Ile Arg Gln Pro Pro Gly Lys Ala Leu Glu 35 40 45 Trp Leu Ala AspIle Trp Trp Asp Asp Lys Lys Asp Tyr Asn Pro Ser 50 55 60 Leu Lys Ser ArgLeu Thr Ile Ser Lys Asp Thr Ser Lys Asn Gln Val 65 70 75 80 Val Leu LysVal Thr Asn Met Asp Pro Ala Asp Thr Ala Thr Tyr Tyr 85 90 95 Cys Ala ArgSer Met Ile Thr Asn Phe Tyr Phe Asp Val Trp Gly Gln 100 105 110 Gly ThrThr Val Thr Val Ser Ser 115 120 25 106 PRT Artificial SequenceDescription of Artificial SequenceAmino acid sequence of light chainvariable region of clone 23 of Figure 7A 25 Asp Ile Gln Met Thr Gln SerPro Ser Thr Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile ThrCys Ser Ala Ser Ser Ser Val Gly Tyr Met 20 25 30 His Trp Tyr Gln Gln LysPro Gly Lys Ala Pro Lys Leu Leu Ile Tyr 35 40 45 Asp Thr Phe Lys Leu AlaSer Gly Val Pro Ser Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Glu PheThr Leu Thr Ile Ser Ser Leu Gln Pro Asp 65 70 75 80 Asp Phe Ala Thr TyrTyr Cys Phe Gln Gly Ser Tyr Tyr Pro Phe Thr 85 90 95 Phe Gly Gly Gly ThrLys Val Glu Ile Lys 100 105 26 120 PRT Artificial Sequence Descriptionof Artificial SequenceAmino acid sequence of heavy chain variable regionof clone 23 of Figure 7B 26 Gln Val Thr Leu Arg Glu Ser Gly Pro Ala LeuVal Lys Pro Thr Gln 1 5 10 15 Thr Leu Thr Leu Thr Cys Thr Phe Ser GlyPhe Ser Leu Ser Thr Ala 20 25 30 Gly Met Ser Val Gly Trp Ile Arg Gln ProPro Gly Lys Ala Leu Glu 35 40 45 Trp Leu Ala Asp Ile Trp Trp Asp Asp LysLys Asp Tyr Asn Pro Ser 50 55 60 Leu Lys Ser Arg Leu Thr Ile Ser Lys AspThr Ser Lys Asn Gln Val 65 70 75 80 Val Leu Lys Val Thr Asn Met Asp ProAla Asp Thr Ala Thr Tyr Tyr 85 90 95 Cys Ala Arg Ser Met Ile Thr Asn PheTyr Phe Asp Val Trp Gly Gln 100 105 110 Gly Thr Thr Val Thr Val Ser Ser115 120

What is claimed is:
 1. A high affinity neutralizing immunoglobulincomprising at least three high affinity complementarity determiningregions (CDRS) wherein each said high affinity CDR has an amino acidsequence selected to result in an immunoglobulin with specificity towardat least one antigenic determinant and having an affinity constant(K_(a)) of at least 10¹⁰M⁻¹ for said antigenic determinant.
 2. The highaffinity neutralizing immunoglobulin of claim 1 wherein saidimmunoglobulin comprises at least four high affinity CDRs.
 3. The highaffinity neutralizing immunoglobulin of claim 1 wherein saidimmunoglobulin has 3 high affinity CDRs.
 4. The high affinityneutralizing immunoglobulin of claim 2 wherein said immunoglobulin has 4high affinity CDRs.
 5. The high affinity neutralizing immunoglobulin ofclaim 2 wherein said affinity constant (K_(a)) is at least 10¹¹.
 6. Thehigh affinity neutralizing immunoglobulin of claim 1 wherein saidimmunoglobulin is specific for at least one protein expressed by avirus.
 7. The high affinity neutralizing immunoglobulin of claim 6wherein said virus is respiratory syncytial virus (RSV).
 8. The highaffinity neutralizing immunoglobulin of claim 7 wherein the protein isthe F protein of RSV.
 9. The high affinity neutralizing immunoglobulinof claim 1 wherein the immunoglobulin has human constant regions. 10.The high affinity neutralizing immunoglobulin of claim 1 wherein theimmunoglobulin binds to the same epitope on RSV as the referenceimmunoglobulin of FIG.
 1. 11. The high affinity neutralizingimmunoglobulin of claim 1 wherein the immunoglobulin includes frameworkderived from only a human immunoglobulin.
 12. The high affinityneutralizing immunoglobulin of claim 1 wherein at least a portion of theframework is derived from a murine immunoglobulin.
 13. The high affinityneutralizing immunoglobulin of claim 1 wherein at least one highaffinity CDR contains a phenylalanine residue at a position where anon-phenylalanine residue occurs in the corresponding CDR of the basicor reference antibody of FIG.
 1. 14. The high affinity neutralizingimmunoglobulin of claim 1 wherein said immunoglobulin comprises a highaffinity H1 CDR and said CDR has an amino acid sequence selected fromthe group consisting of SEQ ID NO: 9 and
 10. 15. The high affinityneutralizing immunoglobulin of claim 1 wherein said immunoglobulincomprises a high affinity H3 CDR having the amino acid sequence of SEQID NO:
 11. 16. The high affinity neutralizing immunoglobulin of claim 1wherein said immunoglobulin comprises a high affinity L2 CDR and saidCDR has an amino acid sequence selected from the group consisting of SEQID NO: 12 and
 13. 19. The high affinity neutralizing immunoglobulin ofclaim 1 wherein said immunoglobulin has a high affinity L3 CDR having anamino acid sequence selected from the group consisting of SEQ ID NO: 14,15 and
 16. 20. The high affinity neutralizing immunoglobulin of claim 1wherein the H1 CDR has the amino acid sequence of SEQ ID NO: 9, the H3CDR has the amino acid sequence of SEQ ID NO: 11, the L2 CDR has theamino acid sequence of SEQ ID NO: 4 and the L3 CDR has the amino acidsequence of SEQ ID NO:
 14. 21. The high affinity neutralizingimmunoglobulin of claim 1 wherein the H1 CDR has the amino acid sequenceof SEQ ID NO: 9, the H3 CDR has the amino acid sequence of SEQ ID NO:11, the L2 CDR has the amino acid sequence of SEQ ID NO: 12 and the L3CDR has the amino acid sequence of SEQ ID NO:
 5. 22. The high affinityneutralizing immunoglobulin of claim 2 wherein the H1 CDR has the aminoacid sequence of SEQ ID NO: 10, the H3 CDR has the amino acid sequenceof SEQ ID NO: 11, the L2 CDR has the amino acid sequence of SEQ ID NO:12 and the L3 CDR has the amino acid sequence of SEQ ID NO:
 14. 23. Thehigh affinity neutralizing immunoglobulin of claim 2 wherein the H1 CDRhas the amino acid sequence of SEQ ID NO: 9, the H3 CDR has the aminoacid sequence of SEQ ID NO: 11, the L2 CDR has the amino acid sequenceof SEQ ID NO: 12 and the L3 CDR has the amino acid sequence of SEQ IDNO:
 14. 24. The high affinity neutralizing immunoglobulin of claim 2wherein the H1 CDR has the amino acid sequence of SEQ ID NO: 9, the H3CDR has the amino acid sequence of SEQ ID NO: 11, the L2 CDR has theamino acid sequence of SEQ ID NO: 12 and the L3 CDR has the amino acidsequence of SEQ ID NO:
 15. 25. The high affinity neutralizingimmunoglobulin of claim 1 wherein the heavy chain variable region hasthe amino acid sequence of SEQ ID NO: 17 and the heavy chain variableregion has the amino acid sequence of SEQ ID NO:
 18. 26. The highaffinity neutralizing immunoglobulin of claim 1 wherein the heavy chainvariable region has the amino acid sequence of SEQ ID NO: 19 and theheavy chain variable region has the amino acid sequence of SEQ ID NO:20.
 27. The high affinity neutralizing immunoglobulin of claim 2 whereinthe heavy chain variable region has the amino acid sequence of SEQ IDNO: 21 and the heavy chain variable region has the amino acid sequenceof SEQ ID NO:
 22. 28. The high affinity neutralizing immunoglobulin ofclaim 2 wherein the heavy chain variable region has the amino acidsequence of SEQ ID NO: 23 and the heavy chain variable region has theamino acid sequence of SEQ ID NO:
 24. 29. The high affinity neutralizingimmunoglobulin of claim 2 wherein the heavy chain variable region hasthe amino acid sequence of SEQ ID NO: 25 and the heavy chain variableregion has the amino acid sequence of SEQ ID NO:
 26. 30. A recombinanthigh affinity neutralizing immunoglobulin having an affinity constant ofat least 10¹⁰ M⁻¹, wherein said immunoglobulin comprises a humanconstant region and a heavy and light chain framework region at leastpart of which is derived from human antibodies.
 31. The recombinant highaffinity neutralizing immunoglobulin of claim 30 wherein the heavy andlight chain framework regions are derived from a consensus sequence ofhuman antibodies.
 32. The recombinant high affinity neutralizingimmunoglobulin of claim 30 wherein the affinity constant is at least10¹¹ M⁻¹.
 33. The recombinant immunoglobulin of claim 32 wherein theheavy and light chain framework regions are derived from a consensussequence of human antibodies.
 34. A composition comprising theimmunoglobulin of claim 1 wherein said immunoglobulin is suspended in apharmacologically acceptable carrier.
 35. A composition comprising theimmunoglobulin of claim 6 wherein said immunoglobulin is suspended in apharmacologically acceptable carrier.
 36. A method of preventing and/ortreating a disease comprising administering to a patient at riskthereof, or afflicted therewith, a therapeutically effective amount ofthe immunoglobulin composition of claim
 34. 37. A method of preventingand/or treating a virus-induced disease comprising administering to apatient at risk thereof, or afflicted therewith, a therapeuticallyeffective amount of the immunoglobulin composition of claim
 35. 38. Amethod of preventing and/or treating respiratory syncytial viruscomprising administering to a patient at risk thereof, or afflictedtherewith, a therapeutically effective amount of the immunoglobulin ofclaim 7 suspended in a pharmaceutically acceptable carrier.
 39. The highaffinity neutralizing immunoglobulin of claim 3 wherein saidimmunoglobulin is selected from the group consisting of Fab, F(ab)′₂, aheavy-light chain dimer, a heavy chain and a light chain.
 40. The highaffinity neutralizing immunoglobulin of claim 4 wherein saidimmunoglobulin is selected from the group consisting of Fab, F(ab)′₂, aheavy-light chain dimer, a heavy chain and a light chain.
 41. The highaffinity neutralizing immunoglobulin of claim 1 wherein saidimmunoglobulin is an antibody.
 42. The high affinity neutralizingimmunoglobulin of claim 2 wherein said immunoglobulin is an antibody.43. The high affinity neutralizing immunoglobulin of claim 6 whereinsaid immunoglobulin is selected from the group consisting of Fab,F(ab)′₂, a heavy-light chain dimer, a heavy chain and a light chain. 44.The high affinity neutralizing immunoglobulin of claim 7 wherein saidimmunoglobulin is selected from the group consisting of Fab, F(ab)′₂, aheavy-light chain dimer, a heavy chain and a light chain.
 45. The highaffinity neutralizing immunoglobulin of claim 8 wherein saidimmunoglobulin is selected from the group consisting of Fab, F(ab)′₂, aheavy-light chain dimer, a heavy chain and a light chain.
 46. A methodof preventing and/or treating a disease comprising administering to apatient at risk thereof, or afflicted therewith, a therapeuticallyeffective amount of the immunoglobulin of claim 39 or 40 suspended in apharmaceutically acceptable carrier.
 47. A method of preventing and/ortreating a virus-induced disease comprising administering to a patientat risk thereof, or afflicted therewith, a therapeutically effectiveamount of the immunoglobulin of claim 43 suspended in a pharmaceuticallyacceptable carrier.
 48. A method of preventing and/or treatingrespiratory syncytial virus comprising administering to a patient atrisk thereof, or afflicted therewith, a therapeutically effective amountof the immunoglobulin of claim 44 or 45 suspended in a pharmaceuticallyacceptable carrier.